Leishmaniasis comprises a group of parasitic illnesses caused by various species of the genus Leishmania. Depending on the immune response of the host, the infectious strain and the virulence of the parasite, the clinical manifestations vary from skin lesions that heal spontaneously, to the visceral (proving fatal if not treated) of the disease. The World Health Organization (WHO) has estimated that some 12 million people worldwide are infected and some 350 million people are at risk of infection (1). In Mediterranean countries the infection is zoonotic and the domestic dog is the main reservoir. The endemic strain of Leishmania in the Mediterranean area is Leishmania infantum, which infects both humans and dogs, producing skin and visceral leishmaniasis (2). Some epidemiological studies have indicated that in Spain approximately 7% of all dogs could be infected, whereas other authors have shown that 10-37% of dogs in the south of France developed visceral leishmaniasis (3). The WHO has estimated that between 2% and 9% of all patients with AIDS in southern Europe developed visceral leishmaniasis, since L. infantum is the cause of the third most frequent parasitic infection in HIV positive patients in the area mentioned (4).
An appropriate method of combating endemic leishmaniasis in Mediterranean countries and other parts of the world would be the creation of a vaccine able to confer long-term immunity against the parasite. Viability of a vaccine against this complex parasite has been suggested by the fact that the patients who recovered from the natural infection developed a strong immunity to Leishmania (5, 6).
As is well known, in mice, dogs and humans, clones of the CD4+ T helper cells (adjuvant) can divide into two functional sub-series, Th1 y Th2, in accordance with the profile of the lymphokines produced. The sub-series Th1 preferentially produces gamma interferon (IFN-γ), whereas the sub-series Th2 produces predominantly the interleukin (IL) 4 [IL-4] (7-9).
In a mouse model infected with Leishmania major (the species that produces skin leishmaniasis), a clear correlation has been found between resistance to infection and the creation of a CD4+Th1 response and, on the other hand, the susceptibility and the development of CD4+Th2 responses (10, 11). Likewise, in humans and dogs resistance to visceral leishmaniasis is also associated with a Th1 response (12-15).
It has recently been shown that interleukin-12 (IL-12) is indispensable to provide protective immunity against L. major, as it initiates protective immune Th1 responses and regulates the proliferation of the subpopulation of T cells (16). It is in fact well established that IL-12 plays a critical role in the generation of Th1 cells and on the optimum differentiation of T cytotoxic lymphocytes (17). On the other hand IL-12 has also been used as a protective adjuvant in other models, such as Schistosoma (25), Listeria (26) or Bordetella (27).
In experimental vaccination tests against murine leishmaniasis, several antigens have been used, achieving different levels of protection. Among these are: gp63 of L. major (18), gp46 (19), LeIF (20, 21), LACK (22) genome libraries of expressing L. major (23). Likewise, the antigen gp46 of L. amazonensis expressed by a recombinant vaccine virus provides encouraging results with a high degree of protection and long-term immunological memory (24).
LACK is a protein of 36 kDa of L. major, so called for its homology with the protein RACK (receiver of activated kinase C in mammals) It has been demonstrated that a fragment of 24 kDa of this protein protects against exposure to L. major in mice when administered subcutaneously as a soluble protein in combination with the co-stimulating cytokine IL-12. Similarly, mice that received this antigen together with IL-12 showed a negative regulation in the number of IL-4 producing cells in the draining lymph nodes 6 weeks after infection with L. major and a positive regulation of the transcripts of IFN-γ in comparison with untreated mice (22). The mice became tolerant to LACK (transgenic mice that expressed the antigen in the thymus). Other studies have shown that the protective efficacy of soluble LACK of L. major together with IL-12 was similar to the efficacy obtained by immunisation with 100 μg of DNA that the same antigen expressed (28).
The protein P 36 of L. infantum has recently been cloned and characterised (29). Analysis of the aminoacid sequence of this protein has shown that P36 is well preserved (96-99%) among the strains studied of Leishmania, L. major and L. chagasi (22, 30).
The protective capacity of P36 of L. infantum has previously been tested in Balb/c mice immunised with this soluble protein or with an expressing system that expresses this protein in some discharge trials that comprised the administration to these animals of an initial dose and another booster dose of the antigen, followed by exposure to L. major promastigotes and it has been determined by the evaluation of the lesions in the pad of the paw where the parasite was inoculated, the parasitic load present in the lymph nodes and the immunological parameters before and after exposure to the parasite and which forms part of the purpose of this patent application PCT [Patent ES200100402, applied for on Feb. 21, 2001 entitled “VACUNA PARA LA PROTECCION DE ANIMALES FRENTE A LEISHMANIA”]. Subsequently, the same authors described for the first time positive results on the protection of the host animal par excellence of the illness, the dog, reservoir in Europe and South America and the type of cellular response against the illness by means of direct infection by L. infantum in dogs. (Patent ES200102057, applied for on 12 Sep. 2001, entitled “ADICION DE LA PATENTE ES200100402 VACUNA PARA LA PROTECCION DE PERROS FRENTE A LEISHMANIA) which is likewise included as part of this Patent application PCT.